Project Summary ? Core B Ex-vivo 3-D ?mini-intestine? culture systems, termed enteroids, generated from human surgical specimens or endoscopic biopsies, have been found to recapitulate the multiple cell types that comprise the crypt-villus axis of the normal intestinal epithelium. The development of these enteroids has provided an exciting opportunity to advance our understanding of how pathogenic as well as beneficial microorganisms, many of which can prevent or lessen the severity of pathogen-induced diarrheal illness, interact with and induce responses from the intestinal epithelium. Therefore, the long range objectives of the Human Enteroid Core are to (1) characterize and compare human enteroids generated from three regions of the small intestine (duodenum, jejunum, ileum) and colon, from different patients to establish their use as models of human diarrheal diseases, (2) test new approaches to improve the enteroids as a relevant model of normal intestinal epithelium, and (3) to provide these enteroids and specialized growth reagents for use in the other projects (Core C, Projects 1, 2, and 3) proposed in this application. The goal of the Human Enteroid Core is to provide a centralized facility to meet the enteroid needs of all of the investigators involved in the NAMSED proposal. The specific functions of the Core will have two functions: a Service component, which will (1) Produce enteroid cultures from our existing bank for all projects (2) Establish enteroids from select new patient samples (3) Maintain frozen stocks of all enteroids (4) Passage and differentiate enteroids per requests (5) Maintain WNT, noggin, and r-spondin producing cells lines and standardized conditioned media for enteroid culture (6) Routinely test the enteroid lines for differentiation status (7) Technology transfer through training group members; a Development component, which will (1) Develop and standardize physiological measures as indicators of enteroid responses (2) Modify enteroids to enhance or deplete the presence of specific cell types or genes (3) Co-culture with mesenchymal cells to test for substitution of growth factors and develop novel structural components (e.g., inversion of the enteroids such that the apical surface faces outward) (4) Incorporate immune cells to improve the relevance of the enteroid model. The Core will be responsive to needs of the individual Projects, which may change as the research projects proceed, as the overall field evolves and as new platforms are engineered in Project 3. New activities will be developed to meet the needs of our and other NASMED project investigators. Our goal is complementary and collaborative in these exciting efforts to develop the enteroids as models to study enteric disease.